Analysis of samples by mass spectrometry often involves the use of one or more stages of ion dissociation or fragmentation, referred to as MS/MS for a single stage of ion dissociation or MSn analysis for multiple stages of ion dissociation. The dissociation of ions generated from a sample yields characteristic product ions, and the measured intensities and mass-to-charge ratios (m/z's) of these product ions are useful for structural elucidation, as well as for detecting and/or quantifying targeted analytes with high specificity and sensitivity. Historically, ion dissociation has been most commonly performed in mass spectrometers by collisional fragmentation techniques known variously as collision induced dissociation (CID), collision activated dissociation (CAD), and higher energy collisional dissociation (HCD). These collisional fragmentation techniques, which produce mainly b- and y-type ions during fragmentation of polypeptides, utilize relatively high energy collisions between precursor analyte molecules or ions and a neutral gas such as helium, nitrogen or argon.
Electron transfer dissociation (ETD) is a more recently developed ion-ion reaction technique in mass spectrometry that utilizes radical anions (negatively charged reagent ions or reagent anions) to transfer electrons to sample precursor/product ions that may result in bond cleavage and consequent generation of product ions. Various aspects of ETD are described in: U.S. Pat. No. 7,534,622 by Hunt, et al.; by Coon, et al. (American Society for Mass Spectrometry, 2005, 16, 880-882); by Emory, et al. (Rapid Communications in Mass Spectrometry, 2009, 23 (3), 409-418); and by Syka et al. in U.S. Patent Application No. US20120156792A1, the disclosures of which are incorporated herein by reference. ETD is an especially valuable technique for the analysis of post-translationally modified peptides and proteins, because ETD induces fragmentation mainly along the peptide backbone in a sequence-independent manner and often leaving labile post translational modifications (PTMs) linked to the peptide chain (unlike collisional dissociation methods, which cleave many PTMs off of the peptide). Furthermore, ETD produces primarily c- and z-type product ions that complement the b- and y-type product ions produced by collisional dissociation, increasing sequence coverage and peptide identifications. Herein, the term, “ion-ion reaction” refers to a reaction that occurs between two different ions of opposite polarity in the gas phase.
The kinetics of ion-ion reaction systems is well understood, with the rate constant for ion-ion capture described by equation 1 below:
                              k          c                =                  v          ⁢                                                    π                2                            ⁡                              [                                                                            Z                      1                                        ⁢                                          Z                      2                                        ⁢                                          e                      2                                                                            μ                    ⁢                                                                                  ⁢                                          v                      2                                                                      ]                                      2                                              Equation        ⁢                                  ⁢        1            where v is the relative velocity of the ion-ion pair, Z1 and Z2 are the charges of the reactant species, e is the electrostatic charge of an electron, and μ is the reduced mass of the collision pair. When a large excess of the reagent ions are maintained throughout the course of the reaction, a pseudo first order criterion is met, with the rate of reaction described by:
                                          A            +                    +                      R            -                          ⁢                  →          k                ⁢        Products                            Equation        ⁢                                  ⁢        2                                r        =                              k            ⁢                                          ⌈                                  A                  +                                ⌉                            ⁢                                                          [                              R                -                            ]                                =                                    k              ′                        ⁡                          [                              A                +                            ]                                                          Equation        ⁢                                  ⁢        3            where [A+] and [R−] represent the precursor analyte and reagent concentrations respectively, k represents the rate coefficient for the reaction and k′ represents the pseudo first order rate coefficient for the reaction (k[R−]). Knowledge of the rate coefficient for the reaction system in conjunction with pseudo first order kinetics allows for prediction of the reaction completeness, and truncation of the reaction at a predetermined point that yields the best chemical information, i.e. at the point that affords the highest spectral signal to noise ratio, or best sequence coverage, for example.
When the pseudo first order approximation breaks down, predicting the amount of reaction completeness becomes challenging, as the rates of the individual chemical reactions are continually changing with time. Furthermore, in cases where the analyte precursor is in a large excess to the reagent, or the ion-ion reaction proceeds through enough generations to significantly deplete or completely consume the reagent population, a desirable amount of reaction completeness may be difficult to achieve. This often be the case during ion-ion reactions of precursors found in many top down proteomics experiments when, for example, the precursor is a large polypeptide, and the number of precursor ion charges in a RF ion containment device is high. Hardware modification to instrumentation by increasing the size of a RF electric field ion containment device where the ion-ion reactions proceed is one solution to this problem but is undesirable and not routinely feasible. Accordingly there is a need for a simpler, alternative approach to optimize the analysis of ion-ion reactions in mass spectrometry without having to resort to significant and expensive hardware changes.